Resolution of components in a chromatographic system is achieved by partitioning solutes between two physcially distinct phases that share a common interfacial boundary. These two phases which are designated the "stationary phase" (P.sub.s) and the "mobile phase" (P.sub.m) are arranged in a narrow channel along the flow axis of the mobile phase and are the heart of the separation system. It may be shown that the distribution of solute molecules between these phases is a constant K usually referred to as the partition coefficient. The partition coefficient for a solute is expressed mathematically as K = C.sub.s /C.sub.m, where C.sub.s is the concentration of solute per unit volume of stationary phase and C.sub.m is the concentration of solute per unit volume of mobile phase. It has been shown experimentally that if the K values for two compounds are sufficiently different, resolution will be achieved during passage through the chromatographic system. Maximum resolution is achieved by choosing the two phases that produce the greatest difference in the partition coefficients of solutes.
The stationary phase in the case of ion exchange and hydrophobic chromatography is immobilized on a support matrix. The basis for partitioning in ion exchange chromatography is ionic association of solutes with an anionic or cationic stationary phase bonded to an inert support. On the other hand, hydrophobic stationary phases also show an affinity for many biological molecules. Differential binding of solutes to hydrophobic supports is based on the relative hydrophobicity of solutes.
Carbohydrates have been used extensively as the inert support matrix to which stationary phases are immobilized for the chromatography of proteins, nucleic acids, polysaccharides, lipoproteins, peptides, hormones, and vitamins. Since they are hydrophilic, water causes carbohydrates to swell into a porous matrix into which biological macromolecules may penetrate and be partitioned. The success of carbohydrates as chromatographic supports is based primarily upon: the ability of the carbohydrate to imbibe large quantities of water and swell into a hydrophilic matrix; the chemical stability of the formed hydrophilic matrix and the ease with which it is derivatized with ion exchange groups; and the ability of carbohydrates to stabilize sensitive biological compounds.
The primary disadvantage of the carbohydrate chromatography supports is that the hydrophilic matrix is sensitive to changes in pH, ionic strength, and pressure. Changes in any of these parameters can cause contraction of the matrix and losses in chromatographic efficiency. This problem is particularly acute in high speed analysis where it is desirable to use forced flow and large changes in pH and ionic strength during the development of columns. Operation of carbohydrate columns under such a protocol results in collapse of the support and complete loss of chromatographic efficiency.
High speed analysis at high P.sub.m flow rates and pressures are currently carried out on rigid inorganic supports. Modern high speed chromatography supports are capable of withstanding both pressures and flow rates 100 times greater than carbohydrate columns. It has not been possible, however, to separate many biological compounds (proteins, nucleic acids, and peptides) on these supports due to adsorption and/or denaturation. Hence high porosity glass, for example, must have both internal and external surfaces coated to accomplish the desired end.
This invention seeks to overcome the disadvantages of both inorganic and carbohydrate supports by forming a composite of the two. These supports have the mechanical ability of inorganic supports and the separation characteristics of carbohydrates.
It has heretofore been known that a bonded support can be produced by covalently linking a layer of carbohydrate material to an inorganic support, and this is shown in copending U.S. Patent Applications, Ser. Nos. 447,640 and 537,197, filed Mar. 4, 1974 and Dec. 30, 1974, respectively, by Frederick E. Regnier, entitled "Bonded Carbohydrate Stationary Phases For Chromatography" and assigned to the assignee of the present invention. U.S. Patent Application Ser. No. 447,640 is now abandoned and U.S. Patent Application Ser. No. 537,197 issued on Sept. 28, 1976 as U.S. Pat. No. 3,983,299.
It has also heretofore been known that a polymer might be used in chromatographic apparatus, but such use has been as the stationary phase rather than as a stabilizing or coupling agent. See, for example, U.S. Pat. No. 3,808,125 issued to Robert G. Good on Apr. 30, 1974.